Comparative study on glutathione S-transferase activity, cDNA, and gene expression between malathion susceptible and resistant strains of the tarnished plant bug, Lygus lineolaris

Abstract

Control of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), in cotton in the mid-South relies heavily on pesticides, mainly organophosphates. Continuous and dominant use of chemical sprays has facilitated resistance development in the tarnished plant bug. A natural population in Mississippi with resistance to malathion was studied to examine whether and how glutathione S-transferases (GST) played an important role in the resistance. Bioassays were first conducted to examine synergism of two GST inhibitors. Both ethacrynic acid (EA) and diethyl maleate (DM) effectively abolished resistance and increased malathion toxicity against two resistant strains by more than 2- and 3-fold, whereas incorporation of GST inhibitors did not significantly increase malathion toxicity against a susceptible strain. GST activities were compared in vitro between malathion susceptible and resistant strains by using 1-chloro-2,4-dinitrobenzene as a GST substrate. The resistant strain had significantly higher (1.5-fold) GST activity than the susceptible strain. Up to 99%, 75%, and 85% of the GST activities were inhibited by EA, sulfobromophthalein (SBT), and DM, respectively. The GST activities tended to increase from May to October by 1.76-fold. All three inhibitors significantly suppressed the GST activity to a constant low level over the season. Further examination of GST cDNA indicated that in the coding region only one nucleotide variation was revealed between the susceptible and resistant strain. This variation did not cause a protein sequence change, and an identical amino acid sequence was predicted for both strains. Multiple sequence alignment and phylogenetic analysis showed that the 216-residue GST from the tarnished plant bug was highly similar (up to 68% of amino acid sequence identity) to the GSTs from other insects, which conferred organophosphate resistance. GST gene expression levels were examined using real-time PCR, and the results indicated that GST gene transcripts were elevated in the resistant strain by 1.3-fold.